The zinc finger-Y (ZFY) gene has been identified from the sex determination region of the human Y chromosome, and postulated as the candidate gene for the testis determining factor (TDF). A highly homologous gene (ZFX) has also been identified on the X chromosome. Both ZFY and ZFX sequences are present on the sex chromosomes of other mammals. Expression studies establish that ZFY genes are transcribed at high levels in adult testis, but at minimal levels in somatic tissues and fetal testes. Furthermore, the ZFY and ZFX genes are differentially expressed as discrete sized transcripts in adult gonadal and somatic tissues. These results raise an important question on whether the ZFY gene is the primary sex determining gene and/or a spermatogenic gene in mammals. The main objective of the proposed studies is to elucidate the functions of the Zfy and Zfx genes in testis determination and spermatogenesis. We plan to concentrate our studies on two species, the mouse and the wood lemming. Sex determination in mouse has been well characterized at genetic levels, and many strains with mutations in their sex determination and spermatogenesis are available. The wood lemming contains a mutated X chromosome, designated X*, which induces X*Y animals to develop into females. Preliminary studies showed that Zfx* gene is molecularly distinct from the Zfx gene. We plan to use recombinant DNA methods to characterize the Zfy and Zfx genes in these two species. We will isolate and sequence their tissue-specific full-length cDNAs, to produce the respective recombinant proteins, and to generate the corresponding tissue-specific antibodies. We will elucidate the molecular differences between the wood lemming Zfx and Zfx* genes. We will study the expression of the Zfy and Zfx genes during normal and abnormal embryogenesis and spermatogenesis. We will correlate the expression patterns to their putative roles in these processes. In addition, We plan to verify and define such roles through transgenic mice assays. We will generate transgenic mice harboring various forms of altered Zfy and Zfx genes, and also attempt to disrupt the normal functions of the endogenous genes through gene targeting and other means. We hope to observe abnormal sex determination and/or spermatogenic activities in the transgenic animals. We believe the proposed studies of the Zfy and Zfx genes in terms of their gene and protein structures and their expression patterns in normal, abnormal and transgenic animals will provide significant information on their probable roles in mammalian testis determination, differentiation and spermatogenesis. This information will be important in the clinical evaluation of gonadal dysgenesis and infertility in man.